Water insoluble polymer: starch-based film coatings for colon targeting

ABSTRACT

A controlled release delivery dosage form for controlled release of an active ingredient, includes an active ingredient coated in a polymeric mixture of: at least a water insoluble polymer and a starch composition including at least one component selected from the group consisting of a starch having an amylose content of between 20 and 45%, a modified starch having an amylose content of between 50 and 80% and a legume starch. The present invention also relates to the use and method for making the same.

FIELD OF THE INVENTION

The present invention relates to a dosage form for the controlleddelivery of active ingredient(s). The present invention also relates tothe use and method for making the same.

BACKGROUND OF THE INVENTION

Colon targeting can be very helpful for many pharmaco-therapies,including the treatment of inflammatory bowel diseases, such as Crohn'sDisease (CD) and Ulcerative Colitis (UC).

If a locally acting drug is orally administered using a conventionalpharmaceutical dosage form, the latter rapidly dissolves in the contentsof the stomach, the drug is released and likely to be absorbed into theblood stream. This leads to elevated systemic drug concentrations and,thus, an increased risk of undesired side effects and at the same timeto low drug concentrations at the site of action in the colon, resultingin poor therapeutic efficiency. These restrictions can be overcome ifdrug release is suppressed in the stomach and small intestine andtime-controlled in the colon. This type of site-specific drug deliveryto the colon might also offer an interesting opportunity for protein andpeptide drugs to get absorbed into the systemic circulation upon oraladministration.

To allow for colon targeting, the drug can for instance be embeddedwithin a polymeric matrix former, or can be drug-loaded tablets orpellets such as spherical beads, approximately 0.5-1 mm in diameter; orcan be coated with a polymeric film. In the upper gastro intestinaltract (GIT), the permeability of the polymeric networks for the drugshould be low, whereas the macromolecular barriers must become permeableonce the colon is reached. This increase in drug permeability of thepolymeric networks at the site of action might be induced by: (i) achange in the pH of the contents of the GIT, (ii) a change in thequality and/or quantity of enzymes along the GIT, or (iii) significantstructural changes within the dosage form occurring after apre-determined lag-time (e.g. crack formation in poorly permeable filmcoatings providing pulsatile drug release patterns).’38 Alternatively,drug release might already start in the stomach and continue throughoutthe GIT, at a rate that is sufficiently low to assure that drug is stillinside the dosage form once the colon is reached.

An attempt to solve the problem of colon targeting is disclosed inUS2005220861A that relates to a controlled release formulation fordelivery of prednisolone sodium metasulphobenzoate. The formulationcomprises prednisolone sodium metasulphobenzoate surrounded by a coatingcomprising glassy amylose, ethyl cellulose and dibutyl sebacate, whereinthe ratio of amylose to ethyl cellulose is from (1:3.5) to (1:4.5) andwherein the amylose is corn or maize amylose. In contrast to theAmerican patent application number US2005220861, the system described inthe present invention is adapted to the disease state of patients. Thisis a very crucial aspect, because to allow for colon targeting thedosage form must become more permeable for the drug once the colon isreached. This can for instance be assured by a preferential degradationof a compound that hinders rapid drug release in the upper gastrointestinal tract. This site-specific degradation can be based onsignificant differences in the quality and quantity of enzymes presentin the upper gastro intestinal tract versus the colon. The compoundshould not be degraded in the upper gastro intestinal tract (and hinderdrug release), but should be degraded in the colon (and, thus, allow fordrug release). The performance of this type of advanced drug deliverysystems is fundamentally depending on the environmental conditions inthe colon of the patients, in particular on the types and concentrationsof the enzymes present in the colon. It is well known and has been welldocumented in the literature that the disease state can significantlyaffect the quality and quantity of the enzyme secreting microflora inthe gastro intestinal tract. This is particularly true for themicroflora in the colon of patients suffering from inflammatory boweldiseases: the quality and quantity of the enzymes present in the colonof a patient can, thus, significantly vary from those in a healthysubject. Consequently, the performance of this type of drug deliverysystems can significantly be affected by the disease state. Systems thatare based on the preferential degradation by enzymes which are notpresent in sufficient concentrations in the disease sate in the colon ofthe patient fail. The present invention reports for the first time ondosage forms allowing for controlled delivery of active ingredient underpathophysiological conditions: in feces of patients suffering frominflammatory bowel diseases. Thus, the performance of these dosage formsis assured under the given pathophysiological conditions in vivo. Thisis decisive for the success and safety of the treatment.

U.S. Pat. No. 6,534,549 relates to a method for producing a controlledrelease composition comprising a mixture of a substantiallywater-insoluble film-forming polymer and amylose in a solvent systemcomprising (1) water and (2) a water-miscible organic solvent which onits own is capable of dissolving the film-forming polymer is contactedwith an active material and the resulting composition dried. Thecomposition is particularly suitable for delivering therapeutic agentsto the colon. In contrast to the present invention that disclosureaddresses drug delivery systems prepared using an organic solvent. Thisis not the case in the present invention. The use of organic solventsimplies several concerns, including toxicity and environmental concernsas well as explosion hazards. Furthermore, the use of amylose impliesthe extraction of this polymer and its stabilization. Amylose isextracted from starch after an hydrolysis and a purification step. Thisprocess is complex and difficulty usable at an industrial level. Thisformulation doesn't take into account the drug release kinetics forpatients suffering from inflammatory bowel diseases. It has to bepointed out that the types and amounts of bacteria present in the colonof inflammatory bowel disease patients can significantly differ fromthose in healthy subjects. Thus, the types and amounts of enzymessecreted by these bacteria and being in contact with the drug deliverysystem can significantly differ. Consequently, the performance of thedrug delivery system can significantly differ.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a delivery dosage formto control the rate and extent of delivery of an active ingredient, forexample, without limitation, an active pharmaceutical ingredient,biological, chemical, nutraceutical, agricultural or nutritional activeingredients.

Another object of the present invention is to provide new polymeric filmcoatings that allow for site-specific drug targeting to the colon andthat may be used for patients suffering from inflammatory bowel diseasesas well as for patients with a healthy colon.

A further object of the present invention is to provide new polymericfilm coatings having a sufficient mechanical stability to withstand theshear stress they are exposed to in the upper GIT (due to the gastrointestinal motility) and to withstand the potentially significanthydrostatic pressure developed within the dosage forms due to waterpenetration into the systems upon contact with aqueous media. Indeed,with known polymer coatings, the problem of accidental crack formationcan result in premature drug release through water-filled channels.

A further object of the present invention is to provide new polymericfilm coatings adjustable to the specific needs of a particular type ofdrug treatment e.g, osmotic activity of the drug and administered dose.

The present invention provides a controlled release delivery dosage formfor controlled release of an active ingredient, comprising an activeingredient coated in a polymeric mixture of:

-   -   at least a water insoluble polymer and    -   a starch composition comprising at least one component selected        from the group consisting of a starch having an amylose content        of between 20 and 45%, preferably of between 25 and 44%, and        more preferably still of between 30 and 40%, a modified starch        having an amylose content of between 50 and 80% and a legume        starch.

Preferentially the controlled release dosage form is an oral formulationand has a gastric resistance. In a preferred embodiment, the controlledrelease pharmaceutical dosage form is in a solid, liquid or semi-liquidform. Advantageously the controlled release pharmaceutical dosage formis a solid dispersion. According to the invention, the polymeric mixtureis an intimate mix of the water insoluble polymer and the starchcomposition, said starch does not form particulates in the waterinsoluble polymer.

In an embodiment of the present invention, the polymeric mixture of thecontrolled release delivery dosage form is a coating mixture, thecontrolled release delivery dosage form comprising a core, the activeingredient being dispersed or dissolved in the core and/or in thecoating mixture.

In a further embodiment of the present invention, the starchcomposition:water insoluble polymer ratio in the controlled releasedelivery dosage form is between 1:2 and 1:8, preferentially 1:3 to 1:6,and more preferentially 1:4 to 1:5.

Preferably, the starch composition has a starch content of at least 50%,preferably of between 70 to 100% more preferably 70 to 100%, still morepreferably between 90 to 100%.

Typically, the starch composition exhibits an amylose content of between20 and 45%, preferentially an amylose content of between 25 and 44%,more preferentially of between 32 and 40%, this percentage beingexpressed in dry weight with respect to the dry weight of starch presentin said composition.

In a further embodiment of the invention, the starch compositioncomprises at least one legume or cereal starch.

Preferably, the legume is selected from the group consisting of pea,bean, broad bean and horse bean.

According to another advantageous alternative form, the legume is aplant, for example a variety of pea or of horse bean, giving seedscomprising at least 25%, preferably at least 40%, by weight of starch(dry/dry). Preferably, the legume starch is a granular legume starch.

Advantageously, the legume is pea. Pea starch granules have twoparticularities. The first one is large granule diameter, larger thanfor example corn starch granules, improving the granule's surface areaand thus contacts with water and micro flora enzymes in the colon. Inaddition, pea starch granules have a high swollen ability improvingtheir surface area thus granules granules digestibility and consequentlythe active ingredient release in the colon.

According to another advantageous alternative the legume starch is anative legume starch.

Advantageously, this starch content of the starch composition is greaterthan 90% (dry/dry). It can in particular be greater than 95%,preferentially greater than 98%.

According to the invention, the modified starch is preferablystabilized. Indeed, according to a preferred embodiment of theinvention, the chemical treatments, which are particularly well suitedto the preparation of a film-forming composition, are the “stabilizing”treatments. Common stabilization modifications may be accomplished byesterifying or etherifying some of the hydroxyl groups along the starchchain. Preferentially, said modified starch is hydroxypropylated and/oracetylated; it being possible for these treatments optionally to besupplemented by a fluidification that is a chemical or enzymatichydrolysis treatment. Preferably, said modified starch isfluidification-treated, for example by acid treatment. The starchcomposition according to the invention thus advantageously comprises atleast one stabilized starch and preferably a hydroxypropylated starchexhibiting a degree of substitution (DS) of at most 0.2. The term “DS”is understood to mean, in the present invention, the mean number ofhydroxypropyl groups per 10 anhydroglucose units. This mean number isdetermined by the standard analytical methods well known to a personskilled in the art.

In a further embodiment of the invention, the starch composition mayadditionally comprise at least one indigestible polysaccharide selectedfrom the group consisting of xylooligosaccharides, inulin,oligofructoses, fructo-oligosacharides (FOS), lactulose, galactomannanand suitable hydrolysates thereof, indigestible polydextrose,indigestible dextrin and partial hydrolysates thereof,trans-galacto-oligosaccharides (GOS), xylo-oligosaccharides (XOS),acemannan, lentinan or beta-glucan and partial hydrolysates thereof,polysaccharides-K (PSK), and indigestible maltodextrin and partialhydrolysates thereof, preferably an indigestible dextrin or anindigestible maltodextrin.

According to the invention, an indigestible maltodextrin or indigestibledextrin having between 15 and 35% of 1->6 glucoside linkages, a reducingsugar content of less than 20%, a polymolecularity index of less than 5and a number-average molecular mass Mn at most equal to 4500 g/mol.

According to a variant, all or some of the said indigestiblemaltodextrins are hydrogenated.

According to a variant, the core has a coating level of 5% to 30%,preferably of 10% to 20%.

In a further embodiment, the polymeric mixture comprises a plasticizer.Preferably the plasticizer content is between 25% to 30% w/w referred tothe water insoluble polymer content.

Preferably, the water insoluble polymer is selected from the groupconsisting of ethyl cellulose, cellulose derivatives, acrylic and/ormethacrylic ester polymers, polymers or copolymers of acrylate ormethacrylate polyvinyl esters, starch derivatives, polyvinyl acetates,polyacrylic acid esters, butadiene styrene copolymers methacrylate estercopolymers, cellulose acetate phtalate, polyvinyl acetate phtalate,shellac, methacrylic acid copolymers, cellulose acetate trimellitate,hydroxypropyl methylcellulose phtalate, zein, starch acetate.

According to a further embodiment the plasticizer is a water solubleplasticizer. Preferably the water soluble plasticizer is selected fromthe group consisting of polyols (glycerin, propylene glycol,polyethylene glycols), organic esters (phtalate esters, dibutylsebacate, citrate esters, triacetin), oils/glycerides (castor oil,acetylated monoglycerides, fractionated coconut oil), soya lecithinalone or as a mixture with one another.

In a preferred embodiment, the controlled release delivery dosage formis a multiparticulate dosage form.

The present invention also provides a method for preparing a controlledrelease delivery dosage form for controlled release of an activeingredient in the colon of patients having a colonic microfloraimbalance or in the colon of healthy subjects, as claimed in claims 1 to12, said method comprising:

-   -   forming a polymeric mixture of:        -   at least one water insoluble polymer and        -   a starch composition comprising at least one component            selected from the group consisting of a starch having an            amylose content of between 20 and 45%, preferably of between            25 and 44%, and more preferably still of between 30 and 40%,            modified starches, a modified starch having an amylose            content of between 50 and 80% and a legume starch.    -   coating said active ingredient in the polymeric mixture.

In a further embodiment, the step of coating the active ingredient is acoating step of a core, the active ingredient being dispersed ordissolved in the core and/or the step of coating the active ingredientis a step of dispersing or dissolving the active ingredient in thepolymeric mixture.

The conditions in the gastro intestinal tract of patients suffering frominflammatory bowel diseases (e.g. Crohn's Diseases and UlcerativeColitis) can significantly differ from those in a healthy subject. Theintra- and inter-individual variability can be substantial with respectto the pH of the GIT contents, types and concentrations ofenzyme-secreting bacteria as well as to the transit times within thevarious GIT segments. For instance, considerable amounts ofbifidobacteria are generally present in the colon of healthy subjectsand are able to degrade complex polysaccharides due to multipleextracellular glycosidases. However, in the disease state theirconcentration can be significantly reduced.’ For example, it was shownthat the fecal glycosidase activity (especially that ofβ-D-galactosidase) is decreased in patients suffering from Crohn'sDisease and that the metabolic activity of the colonic flora is stronglydisturbed in the active disease state.’ Thus, the impact of thepathophysiology can be crucial and can lead to the failure of thepharmaco-treatment.

To avoid treatment failures for patients suffering from inflammatorybowel diseases, the site-specific drug delivery system must be adaptedto the conditions given in the patients' colon. For instance, polymericfilm coatings might be used that are degraded by enzymes, which arepresent in the feces of Crohn's Disease and Ulcerative Colitis patientsin sufficient amounts. However, yet it is unclear which type(s) ofpolymers fulfills these pre-requisites.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Water content of thin films consisting of different types ofpolymer blends (indicated in the figures) upon exposure to: (a) 0.1 MHCl, and (b) phosphate buffer pH 6.8. Films consisting only ofplasticized ethylcellulose are shown for reasons of comparison.

FIG. 2: Dry mass of thin films consisting of different types of polymerblends (indicated in the figures) upon exposure to: (a) 0.1 M HCl, and(b) phosphate buffer pH 6.8. Films consisting only of plasticizedethylcellulose are shown for reasons of comparison.

FIG. 3: Water content and dry mass of thin films consisting of branchedmaltodextrin or peas starch blended with ethylcellulose upon exposure tophosphate buffer pH 6.8 containing or not pancreatin or extract from ratintestine.

FIG. 4: Water content and dry mass of thin films consisting of differenttypes of polysaccharides blended with ethylcellulose upon exposure toculture medium, culture medium inoculated with feces of healthy subjectsand culture medium inoculated with feces of Crohn's Disease (CD)patients and Ulcerative Colitis (UC) patients (as indicated in thefigures). Films consisting only of plasticized ethylcellulose are shownfor reasons of comparison.

FIG. 5: Pictures of the microflora developed upon incubation of thin,polymeric films of different composition (indicated in the figure) withfecal samples of inflammatory bowel disease patients.

FIG. 6: Water uptake and dry mass loss of thin films consisting of PSHP-PG:ethylcellulose blends upon exposure to 0.1 M HCl and phosphatebuffer pH 6.8, respectively. The polymer blend ratio is indicated in thefigures. For reasons of comparison also the behaviour of pure(plasticized) ethylcellulose films is shown.

FIG. 7: Changes in the energy at break of thin PS HP-PG:ethylcellulosefilms upon exposure to: (a) 0.1 M HCl and (b) phosphate buffer pH 6.8.The polymer blend ratio is indicated in the figures. For reasons ofcomparison also the results obtained with pure (plasticized)ethylcellulose films are shown.

FIG. 8: Water uptake and dry mass loss of thin films consisting ofEURYLON® 7 A-PG: ethylcellulose blends upon exposure to 0.1 M HCl andphosphate buffer pH 6.8, respectively. The polymer blend ratio isindicated in the figures. For reasons of comparison also the behaviourof pure (plasticized) ethylcellulose films is shown.

FIG. 9: Changes in the energy at break of thin EURYLON® 7A-PG:ethylcellulose films upon exposure to: (a) 0.1 M HCl and (b)phosphate buffer pH 6.8. The polymer blend ratio is indicated in thefigures. For reasons of comparison also the results obtained with pure(plasticized) ethylcellulose films are shown.

FIG. 10: Water uptake and dry mass loss of thin films consisting ofEURYLON® 6 A-PG:ethylcellulose blends upon exposure to 0.1 M HCl andphosphate buffer pH 6.8, respectively. The polymer blend ratio isindicated in the figures. For reasons of comparison also the behaviourof pure (plasticized) ethylcellulose films is shown.

FIG. 11: Changes in the energy at break of thin EURYLON® 6 A-PG:ethylcellulose films upon exposure to: (a) 0.1 M HCl and (b) phosphatebuffer pH 6.8. The polymer blend ratio is indicated in the figures. Forreasons of comparison also the results obtained with pure (plasticized)ethylcellulose films are shown.

FIG. 12: Water uptake and dry mass loss of thin films consisting ofEURYLON® 6 HP-PG: ethylcellulose blends upon exposure to 0.1 M HCl andphosphate buffer pH 6.8, respectively. The polymer blend ratio isindicated in the figures. For reasons of comparison also the behaviourof pure (plasticized) ethylcellulose films is shown.

FIG. 13: Changes in the energy at break of thin EURYLON® 6 HP-PG:ethylcellulose films upon exposure to: (a) 0.1 M HCl and (b) phosphatebuffer pH 6.8. The polymer blend ratio is indicated in the figures. Forreasons of comparison also the results obtained with pure (plasticized)ethylcellulose films are shown.

FIG. 14: Water uptake kinetics of thin peas starch: ethylcellulose filmsupon exposure to: (a) 0.1 M HCl, and (b) phosphate buffer pH 6.8. Thepolymer:polymer blend ratio (w:w) is indicated in the diagrams.

FIG. 15: Dry mass loss kinetics of thin peas starch: ethylcellulosefilms upon exposure to: (a) 0.1 M HCl, and (b) phosphate buffer pH 6.8.The polymer:polymer blend ratio (w:w) is indicated in the diagrams.

FIG. 16: Mechanical properties of thin peas starch: ethylcellulose filmsin the dry state: (a) puncture strength at break, (b) % elongation atbreak, and (c) energy at break. The polymer:polymer blend ratio (w:w) isindicated on the x-axes.

FIG. 17: Changes in the energy at break of thin peas starch:ethylcellulose films upon exposure to: (a) 0.1 M HCl (for 2 h), or (b)phosphate buffer pH 6.8 (for 8 h) at 37° C. The polymer:polymer blendratio (w:w) is indicated in the diagrams.

FIG. 18: Drug release from pellets coated with peasstarch:ethylcellulose 1:2 under conditions simulating the transitthrough the upper gastro intestinal tract: 2 h exposure to 0.1 M HCl,followed by 9 h exposure to phosphate buffer pH 6.8. The coating levelis indicated in the diagram as well as the absence (solid curves) andpresence (dotted curves) of enzymes (0.32% pepsin at low pH, 1%pancreatin at neutral pH).

FIG. 19: Effects of the peas starch: ethylcellulose blend ratio (w:w)and coating level (indicated in the diagrams) on drug release fromcoated pellets under conditions simulating the transit through the uppergastro intestinal tract: 2 h exposure to 0.1 M HCl, followed by 9 hexposure to phosphate buffer pH 6.8. Solid/dotted curves indicate theabsence/presence of enzymes (0.32% pepsin at low pH, 1% pancreatin atneutral pH).

FIG. 10: Drug release from pellets coated with peasstarch:ethylcellulose 1:4 at a coating level of or 20% (as indicated)under conditions simulating the transit through the entire gastrointestinal tract: 2 h exposure to 0.1 M HCl, followed by 9 h exposure tophosphate buffer pH 6.8, followed by 10 h exposure to: (a) culturemedium inoculated with fresh fecal samples from inflammatory boweldisease patients, or (b) culture medium inoculated with Bifidobacterium.For reasons of comparison, also drug release upon exposure to culturemedium free of fecal samples is shown in (a) (dotted curves).

FIG. 21: Storage stability of pellets coated with 10, 15 and 20% peasstarch:ethylcellulose 1:4: Drug release in 0.1 M HCl (for 2 h) andphosphate buffer pH 6.8 (for 9 h) before (solid curves) and after 1 yearopen storage (dotted curves).

FIG. 22: Macroscopic appearance of the colon of rats, receiving: (A)vehicle only intrarectally (negative control group), (B) TNBSintrarectally (positive control group), (C) TNBS intrarectally and BMD:EC coated pellets orally, or (D) TNBS intrarectally and Pentasa® pelletsorally. TNBS/the vehicle only was administered intrarectally on day 3.The orally administered dose of 5-ASA was 150 mg/kg/day.

FIG. 23: Ameho scores of rats, receiving: (i) TNBS intrarectally, (ii)TNBS intrarectally and Asacol® pellets orally, (iii) TNBS intrarectallyand Pentasa® pellets orally, (iv) TNBS intrarectally and peasstarch:ethylcellulose coated pellets orally, (v) TNBS intrarectally andBMD: ethylcellulose coated pellets orally. TNBS was administeredintrarectally on day 3. The orally administered dose of 5-ASA was 150mg/kg/day.

FIG. 24: Representative histological sections (normal transparietalsection, ×200) of colon tissues of rats. The different layers areindicated: M—mucosa; SM—submucosa; Mu—muscular layer.

Table 1: Concentrations of bacteria [log CFU/g] in the investigatedfecal samples of healthy subjects and inflammatory bowel diseasepatients.

Table 2: Effects of the type of polysaccharide blended withethylcellulose and of the polysaccharide: ethylcellulose blend ratio onthe mechanical properties of thin films in the dry state at roomtemperature.

Table 3: Dissolution media used to simulate the gradual increase in pHalong the GIT.

DETAILED DESCRIPTION OF THE INVENTION

In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions set outherein.

As used herein, the term “active ingredient”, “drug” or“pharmacologically active ingredient” or any other similar term meansany chemical or biological material or compound suitable foradministration by the methods previously known in the art and/or by themethods taught in the present invention, that induces a desiredbiological or pharmacological effect, which may include but is notlimited to (1) having a prophylactic effect on the organism andpreventing an undesired biological effect such as preventing aninfection, (2) alleviating a condition caused by a disease, for example,alleviating pain or inflammation caused as a result of disease, and/or(3) either alleviating, reducing, or completely eliminating the diseasefrom the organism. The effect may be local, such as providing for alocal anaesthetic effect, or it may be systemic.

As used herein, the expression “Colonic microflora imbalance” alsocalled Dysbiosis or dysbacteriosis is intended to mean, in the presentinvention, microbial imbalances as in quality and in quantity in thegastrointestinal tract. This phenomenon is reflected by the quality andquantity of the enzymes present in the colon. Particularly, this alteredmicroflora is observed in the colon of patients suffering frominflammatory bowel diseases, such as Crohn's Disease (CD) and UlcerativeColitis (UC).

As used herein, the term “controlled release delivery” or “controlledrelease” means that the release of the active ingredient out of thedosage form is controlled with respect to time or with respect to thesite of delivery.

The expression “modified starch” should be understood broadly, thisexpression refers for instance to reticulated or acetylated orhydroxypropylated, or more generally to esterification or etherificationstarch.

The term “coat” is used herein to encompass coatings for solid supportsand also capsules enclosing fluids and/or solids and the term “coated”is used similarly.

The expression “water insoluble polymer” should be understood broadly,this expression refers to polymers that do not completely dissolve inwater, such as for example ethyl cellulose, certain starch derivativesor acrylic acid/methacrylic acid derivatives.

The term “indigestible polysaccharides” as used in the present inventionrefers to saccharides which are not or only partially digested in theintestine by the action of acids or digestive enzymes present in thehuman upper digestive tract (small intestine and stomach) but which areat least partially fermented by the human intestinal flora. Indigestiblewater-soluble polysaccharides that may be employed in preferredembodiments of the invention are xylooligosaccharides, inulin,oligofructoses, fructo-oligosacharides (FOS), lactulose, galactomannanand suitable hydrolysates thereof, indigestible polydextrose,indigestible dextrins and partial hydrolysates thereof,trans-galacto-oligosaccharides (GOS), xylo-oligosaccharides (XOS),acemannans, lentinans or beta-glucans and partial hydrolysates thereof,polysaccharides-K (PSK), and indigestible maltodextrins and partialhydrolysates thereof.

Polysaccharide-K is also known as polysaccharide-Krestin (PSK) in Japan,and as polysaccharide-peptide (PS—P) in China. Both have the samechemical and structural characteristics. PSK is a proteoglycan found inthe polypore fungus Trametes versicolor and contains approximately 35%carbohydrate (91% beta-glucan), 35% protein and the remainders are freeresidues such as sugars, amino acids and moisture. PSK is a mixture ofpolysaccharides covalently linked to various peptides with an averagemolecular weight of 100 kilodaltons. The polysaccharide component is ina class of beta-glucans which comprise of glucopyranose units.Structural analysis showed that PSK has a 1, 4-glucan configuration asthe main glucoside portion with branches at positions 3 and 6 at afrequency of one branch per several residual groups of 1-4 bonds.

As used herein, the term “cereal” is intended to mean, in the presentinvention, any plant belonging to the Gramineae, preferably wheat, rice,rye, oats, barley, corn, sorghum and millets.

The term “legume” is intended to mean, in the present invention, anyplant belonging to the Caesalpinaceae, Mimosaceae or Papilionaceaefamilies and in particular any plant belonging to the Papilionaceaefamily, such as, for example, pea, bean, broad bean, horse bean, lentil,alfalfa, clover or lupin.

The expression “starch derivative” means a starch that has beenenzymatically or chemically treated.

The “coating level” means the difference in weight between uncoated andcoated cores that is the weight gain in percentage.

This definition includes in particular all the plants described in anyone of the tables present in the paper by R. Hoover et al. entitled“Composition, Structure, Functionality and Chemical Modification ofLegume Starches: A Review”.

The term “pea” in this instance is considered in its broadest sense andincludes in particular:

-   -   all the wild varieties of smooth pea and    -   all the mutant varieties of smooth pea and of wrinkled pea, this        being the case whatever the uses for which said varieties are        generally intended (food for man, animal nutrition and/or other        uses).

Said mutant varieties are in particular those referred to as “mutantsr”, “mutants rb”, “mutants rug 3”, mutants rug 4″, “mutants rug 5” and“mutants lam” as described in the paper by C-L Heydley et al. entitled“Developing Novel Pea Starches”, Proceedings of the Symposium of theIndustrial Biochemistry and Biotechnology Group of the BiochemicalSociety, 1996, pp. 77-87.

The term “legume starch” is understood to mean any compositionextracted, this being the case in whatever way, from a legume as definedhereinabove and having a starch content of greater than 40%, preferablyof greater than 50% and more preferably still of greater than 75%, thesepercentages being expressed in dry weight with respect to the dry weightof said composition.

Furthermore, it is possible to use starches naturally exhibiting anamylose content within the range selected according to the invention. Inparticular, the starch resulting from legumes may be suitable. Inaccordance with the present invention, this legume starch exhibits anamylose content of less than 45%, more specifically of between 20 20 and45%, preferably of between 25 and 44%, and more preferably still ofbetween 32 and 40%.

For the purpose of the invention, the term “ingestible maltodextrin”means maltodextrin containing indigestible glucosidic linkagesconferring on those maltodextrins additional properties identical todietetic fibers such as “branched maltodextrins”. As used herein, theterm “branched maltodextrins” is intended to mean the ingestiblemaltodextrins described in patent EP 1 006 128, of which the applicantcompany is the proprietor.

According to a preferred variant, said branched maltodextrins have areducing sugar content of between 2% and 5%, and a number-averagemolecular mass Mn of between 2000 and 3000 g/mol.

The branched maltodextrins have a total fiber content of greater than orequal to 50% on a dry basis, determined according to AOAC method No.2001-03 (2001).

The invention provides novel polymeric film coatings for colon targetingwhich are adapted to the disease state of the patients suffering frominflammatory bowel diseases.

Novel polymeric films according to the invention serve as substrates forcolonic bacteria for healthy patients as for patients suffering frominflammatory bowel diseases and are likely to exhibit beneficial effectson the ecosystem of the GIT of the patients. The polymeric film isspecially adapted to the conditions at the target site, also in thedisease state and able to deliver pharmacologically active ingredientsspecifically to the colon.

In the following, the invention will be illustrated by means of thefollowing examples as well as the figures.

Example 1

A. Materials and Methods

A.1. Materials

Branched maltodextrin (BMD) [a branched maltodextrin with non digestibleglycoside linkages: α-1,2 and α-1,3, NUTRIOSE® FB 06 Roquette Fréres],Peas starch (granular pea starch N-735) (35% amylose), a pregelatinizedhydroxypropyl pea starch (PS HP-PG)(LYCOAT® RS 780), a maltodextrin(MD)(GLUCIDEX® 1, Roquette Freres), EURYLON® 7 A-PG (an acetylated andpregelatinized high amylose maize starch (70% amylose) (Roquette Freres,Lestrem, France), EURYLON® 6 A-PG (an acetylated and pregelatinized highamylose maize starch) (60% amylose) (Roquette Freres, Lestrem, France)and EURYLON® 6 HP-PG (a hydroxypropylated and pregelatinized highamylose maize starch (60% amylose) (Roquette Freres, Lestrem, France);aqueous ethylcellulose dispersion (Aquacoat® ECD 30; FMC Biopolymer,Philadelphia, USA); triethylcitrate (TEC; Morflex®, Greensboro, USA);pancreatin (from mammalian pancreas=mixture containing amylase, proteaseand lipase; Fisher Bioblock, Illkirch, France); extract from ratintestine (rat intestinal powder, containing amylase, sucrase,isomaltase and glucosidase; Sigma-Aldrich, Isle d'Abeau Chesnes,France); Columbia blood agar, extracts from beef and yeast as well astryptone (=pancreatic digest of casein) (Becton Dickinson, Sparks, USA);L-cysteine hydrochloride hydrate (Acros Organics, Geel, Belgium);McConkey agar (BioMerieux, Balme-les-Grottes, France); cysteinatedRinger solution (Merck, Darmstadt, Germany).

A.2. Film Preparation

Thin polymeric films were prepared by casting blends of different typesof aqueous polysaccharides and aqueous ethylcellulose dispersion intoTeflon moulds and subsequent drying for 1 d at 60° C. The water solublepolysaccharide was dissolved in purified water (5% w/w) and blended withplasticized ethylcellulose dispersion (25% TEC, overnight stirring; 15%w/w polymer content) at a ratio of 1:3 (polymer:polymer w:w). Themixture was stirred for 6 h prior to casting.

A.3. Film Characterization

The thickness of the films was measured using a thickness gauge(Minitest 600; Erichsen, Hemer, Germany). The mean thickness of allfilms was in the range of 300-340 μm. The water uptake and dry mass losskinetics were measured gravimetrically upon exposure to:

(i) simulated gastric fluid (0.1 M HCl)

(ii) simulated intestinal fluid [phosphate buffer pH 6.8 (USP 30) withor without 1% pancreatin or 0.75% extract from rat intestine]

(iii) culture medium inoculated with feces from healthy subjects

(iv) culture medium inoculated with feces from inflammatory boweldisease patients

(v) culture medium free of feces for reasons of comparison.

Culture medium was prepared by dissolving 1.5 g beef extract, 3 g yeastextract, 5 g tryptone, 2.5 g NaCl and 0.3 g L-cysteine hydrochloridehydrate in 1 L distilled water (pH 7.0±0.2) and subsequent sterilizationin an autoclave. Feces of patients with Crohn's Disease or UlcerativeColitis as well as feces of healthy subjects were diluted 1:200 withcysteinated Ringer solution; 2.5 mL of this suspension was diluted withculture medium to 100 mL. Film pieces of 1.5×5 cm were placed into 120mL glass containers filled with 100 mL pre-heated medium, followed byhorizontal shaking at 37° C. (GFL 3033, Gesellschaft für Labortechnik,Burgwedel, Germany). The incubation with fecal samples was performedunder anaerobic conditions (5% CO₂, 10% H2, 85% N2). At predeterminedtime points samples were withdrawn, excess water removed, the filmsaccurately weighed (wet mass) and dried to constant weight at 60° C.(dry mass). The water content (%) and dry film mass (%) at time t werecalculated as follows:

$\begin{matrix}{{{water}\mspace{14mu} {content}\mspace{14mu} (\%)\mspace{14mu} (t)} = {{\frac{{{wet}\mspace{14mu} {mass}\mspace{14mu} (t)} - {{dry}\mspace{14mu} {mass}\mspace{14mu} (t)}}{{wet}\mspace{14mu} {mass}\mspace{14mu} (t)} \cdot 100}\%}} & (1) \\{{{dry}\mspace{14mu} {film}\mspace{14mu} {mass}\mspace{14mu} (\%)\mspace{14mu} (t)} = {{\frac{{dry}\mspace{14mu} {mass}\mspace{14mu} (t)}{{dry}\mspace{14mu} {mass}\mspace{14mu} \left( {t = 0} \right)} \cdot 100}\%}} & (2)\end{matrix}$

A.4. Bacteriological Analysis

For the bacteriological analysis of fecal samples, the latter werediluted 1:10 with cysteinated Ringer solution. Eight further tenfolddilutions in cysteinated Ringer solution were prepared and 0.1 mL ofeach dilution was plated onto non-selective, modified Columbia bloodagar (for total cultivable counts) and on McConkey agar (being selectivefor enterobacteria). Columbia blood agar plates were incubated during 1week at 37° C. under anaerobic conditions (5% CO2, 10% H2, 85% N2).Colonies were outnumbered; predominant colonies subcultured andidentified based on phenotypic identification criteria. 25 McConkey agarplates were incubated during 48 h at 37° C. in air. The colonies wereoutnumbered and identified using the API 20E system (BioMerieux,Balme-les-Grottes, France). Counts were expressed as log CFU/g (ColonyForming Units per gram) of fresh feces.

For the bacteriological analysis of the microflora developed upon filmincubation with fecal samples, photomicrographs were taken afterGram-staining with an Axiostar plus microscope (Carl Zeiss, Jena,Germany), equipped with a camera (Unit DS-L2, DS camera Head DS-Fi 1;Nikon, Tokyo, Japan). Incubation was performed in a glucides-freeculture medium containing only small amounts of polypeptides (thus,favoring the use of the investigated polysaccharides as substrates)under anaerobic conditions.

B. Results and Discussion

B.1. Film properties in the upper GIT

The permeability of a polymeric system for a drug strongly depends onits water content and dry mass, which determine the density and mobilityof the macromolecules. For instance, in dry hydroxypropylmethylcellulose (HPMC)-based matrix tablets the apparent diffusioncoefficient of a drug approaches zero, whereas in a completely hydratedHPMC gel diffusivities can be reached, which are in the same order ofmagnitude as in aqueous solutions. With increasing water content themacromolecular mobility significantly increases and, thus, the freevolume available for diffusion. In some systems, the polymer undergoes aglassy-to-rubbery phase transition as soon as a critical water contentis reached. This leads to a significant, stepwise increase in polymerand drug mobility. Thus, the water content of a polymeric film coatingcan give important insight into the macromolecular mobility and, hence,permeability for a drug. FIGS. 1 a and 1 b show the water uptakekinetics of thin films consisting of various types of polysaccharide:polysaccharide ethylcellulose blends in 0.1 N HCl and phosphate bufferpH 6.8, respectively. The presence of ethylcellulose in all films allowsavoiding premature dissolution in the upper GIT. The investigatedpolysaccharides are all water-soluble and aim at providing thesensitivity of the coatings' drug permeability to the surroundingenvironment: Once the colon is reached, the polysaccharides are to beenzymatically degraded and drug release to be started. Thepolysaccharide: polysaccharide ethylcellulose blend ratio in FIG. 1 isconstant: 1:3. Clearly, the water uptake rates and extents significantlydepend on the type of polysaccharide. The ideal film coating allowingfor colon targeting should take up only small amounts of water at a lowrate in both media in order to prevent premature drug release in theupper GIT. As it can be seen, blends of ethylcellulose and BMD or peasstarch are most promising for this purpose. Plasticized ethylcellulosefilms without water-soluble polysaccharide take up only minor amounts ofwater (empty circles).

In addition to the water uptake kinetics also the dry mass lossbehaviour of thin polymeric films serves as an indicator for thecoatings' permeability for the drug, and, hence, potential to suppresspremature release within the upper GIT. If the films loose significantamounts of dry mass upon exposure to the release media, the coatings canbe expected to become permeable for many drugs, in particular those witha low molecular weight such as 5-aminosalicylic acid (5-ASA, 153.1 Da).FIGS. 2 a and 2 b illustrate the experimentally determined dry mass lossof thin films consisting of various polysaccharide: polysaccharideethylcellulose blends (constant ratio=1:3) upon exposure to 0.1 N HCland phosphate buffer pH 6.8, respectively. The ideal film looses onlyminor amounts of dry mass at a low rate (or no mass at all), assuringdense polymeric networks which are poorly permeable for the incorporateddrug under these conditions. As it can be seen, the dry mass loss ofpeas starch- and BMD-containing films is very low, even after up to 8 hexposure to these release media. The observed decrease in dry mass canat least partially be attributed to the leaching of the water-solubleplasticizer triethyl citrate (TEC, used to plasticize the aqueousethylcellulose dispersion) into the bulk fluid. In addition, parts ofthe water-soluble polysaccharide might leach out of the films.Plasticized ethylcellulose films without water-soluble polysaccharideloose only very small amounts of water, irrespective of the type ofrelease medium (empty circles). However, the permeability of intactethylcellulose films is known to be very low for many drugs, which canat least partially be attributed to the low water-uptake rates andextents of these systems. For this reason, intact ethylcellulose filmsare also used as moisture protective coatings. Please note that the lossof the water-soluble plasticizer TEC into the bulk fluids can beexpected to be much more pronounced in films containing 25% (w/w)water-soluble polysaccharides compared to pure (plasticized)ethylcellulose films, because the increased water uptake rates andextents (FIG. 1) of the blended systems lead to much higher polymerchain mobility and, thus, also increased TEC mobility.

It has to be pointed out that the results shown in FIG. 2 were obtainedin the absence of any enzymes. It is well known that pancreatic enzymescan degrade certain polysaccharides and, thus, potentially inducesignificant mass loss and water uptake under in vivo conditions,resulting in increased film permeability for the drug. To clarify theimportance of this phenomenon, the water uptake kinetics and dry massloss behaviour of the thin films were also measured in the presence ofpancreatin (=mixture containing amylase, protease and lipase) and of anextract from rat intestine (containing amylase, sucrase, isomaltase andglucosidase) in phosphate buffer pH 6.8 (FIG. 3). Clearly, the additionof these enzymes did not significantly affect the resulting water uptakeand dry mass loss kinetics of the investigated films. Thus, the latterdo not serve as substrates for these enzymes.

B.2. Film Properties in the Colon

Once the colon is reached, the polymeric film coatings should becomepermeable for the drug. This can for instance be induced by (partial)enzymatic degradation. Importantly, the concentrations of certainenzymes are much higher in the colon than in the upper GIT. Thisincludes enzymes, which are produced by the natural microflora of thecolon (this part of the GIT contains much more bacteria than the stomachand small intestine). However, great caution must be paid when usingthis type of colon targeting approach, because the microflora ofpatients suffering from inflammatory bowel diseases can be significantlydifferent from the microflora of healthy subjects. Thus, the drugdelivery system must be adapted to the disease state of the patient.Table 1 shows for instance the concentrations of the bacteria determinedin the fecal samples of the healthy subjects as well as of the Crohn'sDisease and Ulcerative Colitis patients included in this study.Importantly, there were significant differences, in particular withrespect to the concentrations of Bifidobacterium (being able to degradecomplex polysaccharides due to multiple extracellular glycosidases) andEscherichia coli, which where present at much higher concentrations inthe feces of healthy subjects compared to the feces of the inflammatorybowel disease patients. In contrast, the fecal samples of the Crohn'sDisease and Ulcerative Colitis patients contained lactose negative E.coli, Citrobacter freundii, Klebsiella pneumoniae, Klebsiella oxytocaand Enterobacter cloacae, which were not detected in healthy subjects.Thus, there are fundamental differences in the quality and quantity ofthe microflora, which must be taken into account: Polymeric filmcoatings, which allow for colon targeting under physiological conditionsin a healthy volunteer, might fail under the pathophysiologicalconditions in the disease state of a patient. To address this verycrucial point, which is very often neglected, the water uptake and drymass loss of thin films consisting of various types of polysaccharide:polysaccharide ethylcellulose blends were determined upon exposure tofecal samples from Crohn's Disease and Ulcerative Colitis patients aswell as to the feces of healthy subjects and to pure culture medium forreasons of comparison (FIG. 4). Appropriate films should take upconsiderable amounts of water and show significant dry mass loss uponexposure to patients' feces in order to induce drug release at the siteof inflammation in the colon. As it can be seen in FIGS. 4 a and 4 b,films based on ethylcellulose: BMD and ethylcellulose: peas starch(which are the two most promising types of polymer blends based on theabove described results obtained in media simulating the contents of theupper GIT) show significant water uptake and dry mass loss upon exposureto the feces of Crohn's Disease patients, Ulcerative Colitis patients aswell as of healthy subjects. Please note that also other types ofpolymer blends look promising with respect to the presented films' wateruptake and dry mass loss behaviour upon exposure to fecal samples (oreven more appropriate than ethylcellulose: BMD and ethylcellulose: peasstarch blends). However, these systems already take up considerableamounts of water and remarkably loose in dry mass upon contact withmedia simulating the contents of the upper GIT (FIGS. 1 and 2).

The fact that the investigated polymeric films serve as substrates forthe bacteria in feces from inflammatory bowel disease patients could befurther confirmed by the analysis of the microflora developed upon filmexposure to fecal samples under anaerobic conditions at 37° C. (FIG. 5).Clearly, specific types of bacteria proliferated upon incubation withthe blended films. Importantly, this phenomenon can be expected to behighly beneficial for the ecosystem of the GIT of the patients in thedisease state, normalizing the microflora in the colon. This verypositive, pre-biotic effect comes in addition to the drug targetingeffect. Biological samples incubated without any polymeric films or withpure (plasticized) ethylcellulose films showed much less bacterialgrowth (FIG. 5).

The novel polymeric film coatings identified for colon targeting arecomposed of water insoluble polymer: polysaccharide particularlyethylcellulose: BMD, ethylcellulose: pea starch, ethylcellulose: MD,ethylcellulose: EURYLON® 6 A-PG, ethylcellulose: EURYLON® 6 HP-PG andethylcellulose: EURYLON® 7 A-PG blends, which are adapted to the diseasestate of the patients. Importantly, low water uptake and dry mass lossrates and extents in media simulating the contents of the upper GIT canbe combined with elevated water uptake and dry weight loss upon contactwith feces from inflammatory bowel disease patients. Changes in thecomposition of the flora in the colon of patients indicate that thesepolysaccharides serve as substrates for colonic bacteria in the diseasestate and are likely to exhibit beneficial effects on the ecosystem ofthe GIT of the patients. The obtained new knowledge, thus, provides thebasis for the development of novel polymeric film coatings able todeliver drugs specifically to the colon. Importantly, these polymericbarriers are adapted to the conditions at the target site in the diseasestate.

Example 2

A. Materials and Methods

A.1. Materials

Pregelatinized hydroxypropyl pea starch (PS HP-PG) (LYCOAT® RS 780,Roquette Freres), EURYLON® 7 A-PG (an acetylated and pregelatinized highamylose maize starch (70% amylose)) (Roquette Freres, Lestrem, France),EURYLON® 6 A-PG (an acetylated and pregelatinized high amylose maizestarch (60% amylose)) (Roquette Freres, Lestrem, France) and EURYLON® 6HP-PG (a hydroxypropylated and pregelatinized high amylose maize starch(60% amylose)) (Roquette Freres, Lestrem, France); aqueousethylcellulose dispersion (Aquacoat ECD 30; FMC Biopolymer,Philadelphia, USA); triethylcitrate (TEC; Morflex, Greensboro, USA).

A.2. Preparation of thin, polymeric films

Thin polymeric films were prepared by casting blends of different typesof polysaccharides and aqueous ethylcellulose dispersion into Teflonmoulds and subsequent drying for 1 d at 60° C. The water solublepolysaccharide was dissolved in purified water (5% w/w, in the case ofEURYLON® 7 A-PG, EURYLON® 6 A-PG and EURYLON® 6 HP-PG in hot water),blended with plasticized aqueous ethylcellulose dispersion (25.0, 27.5or 30.0% w/w TEC, referred to the ethylcellulose contentovernightstirring; 15% w/w polymer content) at a ratio of 1:2, 1:3, 1:4, 1:5(polymer:polymer w:w), as indicated. The mixtures were stirred for 6 hprior to casting.

A.3. Film Characterization

The thickness of the films was measured using a thickness gauge(Minitest 600; Erichsen, Hemer, Germany). The mean thickness of allfilms was in the range of 300-340 μm. The water uptake and dry mass losskinetics of the films were measured gravimetrically upon exposure to 0.1M HCl and phosphate buffer pH 6.8 (USP 30) as follows: Pieces of 1.5×5cm were placed into 120 mL plastic containers filled with 100 mLpre-heated medium, followed by horizontal shaking at 37° C. (80 rpm, GFL3033; Gesellschaft fuer Labortechnik, Burgwedel, Germany). Atpredetermined time points samples were withdrawn, excess water removed,the films accurately weighed (wet mass) and dried to constant weight at60° C. (dry mass). The water content (%) and dry film mass (%) at time twere calculated as follows:

$\begin{matrix}{{{Water}\mspace{14mu} {content}\mspace{14mu} (\%)\mspace{14mu} (t)} = {{\frac{{{wet}\mspace{14mu} {mass}\mspace{14mu} (t)} - {{dry}\mspace{14mu} {mass}\mspace{14mu} (t)}}{{wet}\mspace{14mu} {mass}\mspace{14mu} (t)} \cdot 100}\%}} & (1) \\{{{Dry}\mspace{14mu} {film}\mspace{14mu} {mass}\mspace{14mu} (\%)\mspace{14mu} (t)} = {{\frac{{dry}\mspace{14mu} {mass}\mspace{14mu} (t)}{{dry}\mspace{14mu} {mass}\mspace{14mu} \left( {t = 0} \right)} \cdot 100}\%}} & (2)\end{matrix}$

A.4. Mechanical Properties of Thin Films

The mechanical properties of the films in the dry and wet state weredetermined with a texture analyzer (TAXT.Plus, Winopal Forschungsbedarf,Ahnsbeck, Germany) and the puncture test. Film specimens were mounted ona film holder (n=6). The puncture probe (spherical end: 5 mm diameter)was fixed on the load cell (5 kg), and driven downward with a cross-headspeed of 0.1 mm/s to the center of the film holder's hole. Load versusdisplacement curves were recorded until rupture of the film and used todetermine the mechanical properties as follows:

$\begin{matrix}{{{Puncture}\mspace{14mu} {strength}} = \frac{F}{A}} & (3)\end{matrix}$

Where F is the load required to puncture the film and A thecross-sectional area of the edge of the film located in the path.

$\begin{matrix}{{\% \mspace{14mu} {elongation}\mspace{14mu} {at}\mspace{14mu} {break}} = {{\frac{\sqrt{R^{2} + D^{2}} - R}{R} \cdot 100}\%}} & (4)\end{matrix}$

Here, R denotes the radius of the film exposed in the cylindrical holeof the holder and D the displacement.

$\begin{matrix}{{{Energy}\mspace{14mu} {at}\mspace{14mu} {break}\mspace{14mu} {unit}\mspace{14mu} {volume}} = \frac{A\; U\; C}{V}} & (5)\end{matrix}$

Where AUC is the area under the load versus displacement curve and V thevolume of the film located in the die cavity of the film holder.

B. Results and Discussion

B.1. PS HP-PG: Ethylcellulose Blends

FIG. 6 shows the gravimetrically determined water uptake and dry massloss kinetics of thin films consisting of different types of PS HP-PG:ethylcellulose blends upon exposure to 0.1 M HCl and phosphate buffer pH6.8, respectively. PS HP-PG is a pregelatinized modified starch. As inthe case of MD, the resulting extent and rate of the water penetrationinto the systems significantly increased when increasing thepolysaccharide:ethylcellulose ratio from 1:5 to 1:2 (FIG. 12, top row).This can again be attributed to the higher hydrophilicity of thepolysaccharide compared to ethylcellulose. Appropriately elevatedcoating levels are likely to be required to suppress the prematurerelease of freely water-soluble, small molecular weight drugs in theupper GIT at high initial PS HP-PG contents. Also the rate and extent ofthe films' dry mass loss significantly increased with increasing PSHP-PG contents, due to partial TEC and polysaccharide leaching. In allcases, the rates and extents of the water penetration and dry mass losswere higher in phosphate buffer pH 6.8 compared to 0.1 M HCl, because ofthe pH-dependent ionization of SDS as discussed above. As in the case ofMD: ethylcellulose blends, the mechanical stability of PSHP-PG:ethylcellulose films could effectively be adjusted by varying theinitial ethylcellulose content. This was true for the puncture strength,% elongation at break and energy at break in the dry state at roomtemperature (Table 2) as well as for the mechanical resistance in thewet sate upon exposure to 0.1 M HCl and phosphate buffer pH 6.8 (FIG.7). The decrease in the energy at break with time can again beattributed to partial plasticizer and polysaccharide leaching into thebulk fluids, irrespective of the type of release medium.

B.2. EURYLON® 7 A-PG: Ethylcellulose Blends

The water uptake and dry mass loss kinetics of thin films consisting of1:2 to 0:1 EURYLON® 7 A-PG: ethylcellulose blends in 0.1 M HCl andphosphate buffer pH 6.8 are shown in FIG. 8. EURYLON® 7 A-PG is anacetylated and pregelatinized high amylose maize starch (70% amylose)(Roquette Freres, Lestrem, France). As it can be seen, the sametendencies as with MD: ethylcellulose and PS HP-PG: ethylcelluloseblends were observed: (i) the water uptake rates and extents increasedwith decreasing ethylcellulose contents, (ii) the dry mass loss ratesand extents increased with increasing polysaccharide contents, (iii)these effects were more pronounced in phosphate buffer pH 6.8 than in0.1 M HCl. Importantly, the water contents of the films upon 2 hexposure to phosphate were considerable: about 50% w/w. Thus, also athigh initial EURYLON® 7 A-PG contents, elevated coating levels arelikely to be required in order to suppress the premature release offreely water-soluble, low molecular weight drugs in the upper GIT.Importantly, the mechanical resistance of the EURYLON® 7 A-PG:ethylcellulose based films was significantly higher than that of filmsconsisting of MD: ethylcellulose and PS HP-PG: ethylcellulose blends inthe dry state at room temperature (Table 2). However, these differencesbecame minor when the films were exposed to 0.1 M HCl and phosphatebuffer pH 6.8, irrespective of the type of release medium (FIG. 9).Importantly, the variation of the polymer blend ratio again allowed foran efficient adjustment of the mechanical stability of the films.

B.3. EURYLON® 6 A-PG: Ethylcellulose and EURYLON® 6 HP-PG:Ethylcellulose Blends

EURYLON® 6 A-PG is an acetylated and pregelatinized high amylose maizestarch (60% amylose) (Roquette Freres, Lestrem, France), and EURYLON® 6HP-PG a hydroxypropylated and pregelatinized high amylose maize starch(60% amylose) (Roquette Freres, Lestrem, France). Interestingly, the drymass loss of thin films consisting of EURYLON® 6 A-PG: ethylcelluloseand EURYLON® 6 HP-PG: ethylcellulose blends was much less pronouncedthan that of the other investigated polymer blends upon exposure to 0.1M HCl and phosphate buffer pH 6.8, respectively (FIGS. 10 and 12, bottomrows). This was true for both, the rates and the extents of the dry massloss and for all the investigated polymer blend ratios. In contrast, thewater uptake rates and extents of these films upon exposure to thedifferent release media were similar to those of the otherpolysaccharide: ethylcellulose blends, reaching water contents ofapproximately 50% w/w after 1-2 h exposure to phosphate buffer pH 6.8 inthe case of high initial polysaccharide contents (FIGS. 10 and 12, toprows). Thus, also for EURYLON® 6 A-PG: ethylcellulose and EURYLON® 6HP-PG: ethylcellulose blends elevated coating levels are likely to berequired to suppress premature release of freely water-soluble, lowmolecular weight drugs in the upper GIT at low initial ethylcellulosecontents. As it can be seen in Table 2, the mechanical properties ofthin films consisting of these types of polymer blends in the dry stateat room temperature are similar to those of EURYLON® 7 A-PG:ethylcellulose blends at the same blend ratios. As in the case of thelatter blends, exposure to 0.1 M HCl or phosphate buffer pH 6.8 resultedin a decrease in the mechanical stability of the macromolecularnetworks, irrespective of the type of release medium and polymer blendratio (FIGS. 11 and 13). Importantly, desired system stabilities canagain effectively be adjusted by varying the polymer blend ratio.

The key properties of thin polymeric films consisting of polysaccharide:water insoluble polymer blends exhibiting an interesting potential toprovide site specific drug delivery to the colon (and being adapted tothe pathophysiology of inflammatory bowel disease patients) caneffectively be adjusted by varying the polymer blend ratio and type ofpolysaccharide. This includes the water uptake and dry mass losskinetics as well as the mechanical properties of the films before andupon exposure to aqueous media simulating the contents of the upper GIT.Thus, broad ranges of film coating properties can easily be provided,being adapted to the needs of the respective drug treatment (e.g.,osmotic activity of the core formulation and administered dose)

Example 3

A. Materials and methods

A.1. Materials

Peas starch N-735 (peas starch; Roquette Freres, Lestrem, France);Aquacoat ECD 30 (aqueous ethylcellulose dispersion; FMC Biopolymer,Brussels, Belgium); triethylcitrat (TEC; Morflex, Greensboro, N.C.,USA); 5-aminosalicylic acid (5-ASA; Sigma-Aldrich, Isle d'Abeau Chesnes,France); microcrystalline cellulose (Avicel PH 101; FMC Biopolymer);bentonite and polyvinylpyrrolidone (PVP, Povidone K 30) (CoopertationPharmaceutique Francaise, Melun, France); pancreatin (from mammalianpancreas=mixture of amylase, protease and lipase) and pepsin (FisherBioblock, Illkirch, France); extracts from beef and yeast as well astryptone (=pancreatic digest of casein) (Becton, Dickinson and Company,Franklin Lakes, N.J., USA); L-cysteine hydrochloride hydrate (AcrosOrganics, Geel, Belgium); cysteinated Ringer solution (Merck, Darmstadt,Germany).

A.2. Preparation of Free Films

Thin, free films were prepared by casting blends of peas starch andaqueous ethylcellulose dispersion (plasticized with 25% TEC) onto Teflonmoulds and subsequent controlled drying (1 d at 60° C.). Peas starch wasdispersed in purified water at 65-75° C. (5% w/w). Aqueousethylcellulose dispersion (15% w/w solids content) was plasticized for24 h with 25% TEC (w/w, referred to the solids content of thedispersion). The peas starch and ethylcellulose dispersions were blendedat room temperature at the following ratios: 1:2, 1:3, 1:4 and 1:5(polymer:polymer, w:w). The mixtures were stirred for 6 h prior tocasting.

A.3. Characterization of Free Films

The thickness of the films was measured using a thickness gauge(Minitest 600; Erichsen, Hemer, Germany). The mean thickness of allfilms was in the range of 300-340 μm.

The water uptake and dry mass loss kinetics of the films were measuredgravimetrically upon exposure to: (i) simulated gastric fluid (0.1 MHCl), and (ii) simulated intestinal fluid [phosphate buffer pH 6.8 (USP32)] at 37° C. as follows: Pieces of 1.5 cm×5 cm were placed into 120 mLplastic containers filled with 100 mL pre-heated medium, followed byhorizontal shaking at 37° C. (80 rpm, GFL 3033; Gesellschaft fuerLabortechnik, Burgwedel, Germany). At predetermined time points sampleswere withdrawn, excess water removed, the films accurately weighed (wetmass) and dried to constant weight at 60° C. (dry mass). The watercontent (%) and dry film mass (%) at time t were calculated as follows:

$\begin{matrix}{{{water}\mspace{14mu} {content}\mspace{14mu} (\%)\mspace{14mu} (t)} = {{\frac{{{wet}\mspace{14mu} {mass}\mspace{14mu} (t)} - {{dry}\mspace{14mu} {mass}\mspace{14mu} (t)}}{{wet}\mspace{14mu} {mass}\mspace{14mu} (t)} \cdot 100}\%}} & (1) \\{{{dry}\mspace{14mu} {film}\mspace{14mu} {mass}\mspace{14mu} (\%)\mspace{14mu} (t)} = {{\frac{{dry}\mspace{14mu} {mass}\mspace{14mu} (t)}{{dry}\mspace{14mu} {mass}\mspace{14mu} \left( {t = 0} \right)} \cdot 100}\%}} & (2)\end{matrix}$

The mechanical properties of the films were determined using a textureanalyzer (TAXT.Plus; Winopal Forschungsbedarf, Ahnsbeck, Germany) andthe puncture test in the dry state and upon exposure to 0.1 M HCl andphosphate buffer pH 6.8 (in the wet state). Film specimens were mountedon a film holder (n=6). The puncture probe (spherical end: 5 mmdiameter) was fixed on the load cell (5 kg), and driven downward with across-head speed of 0.1 mm/s to the center of the film holder's hole.Load versus displacement curves were recorded until rupture of the filmand used to determine the mechanical properties as follows:

$\begin{matrix}{{{puncture}\mspace{14mu} {strength}\mspace{14mu} {at}\mspace{14mu} {break}} = \frac{F}{A}} & (3)\end{matrix}$

where F is the load required to puncture the film and A thecross-sectional area of the edge of the film located in the path.

$\begin{matrix}{{\% \mspace{14mu} {elongation}\mspace{14mu} {at}\mspace{14mu} {break}} = {{\frac{\sqrt{R^{2} + D^{2}} - R}{R} \cdot 100}\%}} & (4)\end{matrix}$

Here, R denotes the radius of the film exposed in the cylindrical holeof the holder and D the displacement.

$\begin{matrix}{{{energy}\mspace{14mu} {at}\mspace{14mu} {break}\mspace{14mu} {per}\mspace{14mu} {unit}\mspace{14mu} {volume}} = \frac{A\; U\; C}{V}} & (5)\end{matrix}$

Where AUC is the area under the load versus displacement curve and V thevolume of the film located in the die cavity of the film holder.

A.4. Preparation of Coated Pellets

Drug (5-amino salicylic acid, 5-ASA) loaded pellet starter cores(diameter: 0.7-1.0 mm; 60% 5-ASA, 32% microcrystalline cellulose, 4%bentonite, 4% PVP) were prepared by extrusion and subsequentspheronisation as follows: The respective powders were blended in a highspeed granulator (Gral 10; Collette, Antwerp, Belgium) and purifiedwater was added until a homogeneous mass was obtained (41 g of water for100 g of powder blend). The wetted mixture was passed through a cylinderextruder (SK M/R, holes: 1 mm diameter, 3 mm thickness, rotation speed:96 rpm; Alexanderwerk, Remscheid, Germany). The extrudates weresubsequently spheronised at 520 rpm for 2 min (Spheroniser Model 15;Calveva, Dorset, UK) and dried in a fluidized bed (ST 15; Aeromatic,Muttenz, Switzerland) at 40° C. for 30 min. The size fraction 0.7-1.0 mmwas obtained by sieving. These drug loaded starter cores were thencoated in a fluidized bed coater, equipped with a Wurster insert (Strea1; Aeromatic-Fielder, Bubendorf, Switzerland) with different peasstarch: ethylcellulose blends until a weight gain of 5, 10, 15 or 20%(w/w) was achieved. The coating formulations were prepared in the sameway as the dispersions used for film casting (as described in section2.2. Preparation of free films). The process parameters were as follows:inlet temperature=39±2° C., product temperature=40±2° C., sprayrat=1.5-3 g/min, atomization pressure=1.2 bar, nozzle diameter=1.2 mm.Afterwards, the pellets were further fluidized for 10 min andsubsequently cured in an oven for 24 h at 60° C.

A.5. Drug Release from Coated Pellets

Drug release from coated pellets was measured in media simulating theconditions in the:

Upper gastro intestinal tract: Pellets were placed into 120 mL plasticcontainers, filled with 100 mL dissolution medium: 0.1 M HCl (optionallycontaining 0.32% pepsin) during the first 2 h, and phosphate buffer pH6.8 (USP 32) (optionally containing 1% pancreatin) during the subsequent9 h. The flasks were agitated in a horizontal shaker (80 rpm; GFL 3033).At pre-determined time points, 3 mL samples were withdrawn and analyzedUV-spectrophotometrically for their drug content (A=302.6 nm in 0.1 MHCl; λ=330.6 nm in phosphate buffer pH 6.8) (UV-1650; Shimadzu, Champssur Marne, France). In the presence of enzymes, the samples werecentrifuged for 15 min at 11000 rpm and subsequently filtered (0.2 μm)prior to UV-measurements. Each experiment was conducted in triplicate.

Entire gastro intestinal tract: Pellets were exposed to 0.1 M HCl for 2h and subsequently to phosphate buffer pH 6.8 (USP 32) for 9 h in a USPApparatus 3 (Bio-Dis; Varian, Paris, France) (dipping speed=10 dpm).Afterwards, the pellets were transferred into 120 mL flasks filled with:(i) 100 mL culture medium inoculated with feces from inflammatory boweldisease patients, (ii) culture medium inoculated with Bifidobacterium,or (iii) culture medium free of feces and bacteria for reasons ofcomparison. The samples were agitated (50 rpm) at 37° C. under anaerobicconditions (5% CO2, 10% H2, 85% N2). Culture medium was prepared bydissolving 1.5 g beef extract, 3 g yeast extract, 5 g tryptone, 2.5 gNaCl and 0.3 g L-cysteine hydrochloride hydrate in 1 L distilled water(pH 7.0±0.2) and subsequent sterilization in an autoclave. Feces ofpatients suffering from Crohn's disease or ulcerative colitis werediluted 1:200 with cysteinated Ringer solution; 2.5 mL of thissuspension was diluted with culture medium to 100 mL. At pre-determinedtime points, 2 mL samples were withdrawn, centrifuged at 13000 rpm for 5min, filtered (0.22 μm) and analyzed by HPLC for their drug content(ProStar 230; Varian). The mobile phase consisted of 10% methanol and90% of an aqueous acetic acid solution (1% w/v) (Siew et al., 2000a).Samples were injected into a Pursuit C18 column (150×4.6 mm; 5 μm), theflow rate was 1.5 mL/min. The drug was detectedUV-spectrophotometrically at λ=300 nm.

Drug release was measured from freshly prepared pellets (if nototherwise stated), as well as from pellets stored for 1 year at roomtemperature (23±2° C.) and ambient relative humidity (55±5%) in openglass vials.

B. Results and Discussion

B.1. Water Uptake and Dry Mass Loss of Thin Films

Ideally, a polymeric film coating allowing for site specific drugdelivery to the colon should effectively suppress drug release in theupper part of the gastro intestinal tract: the stomach and the smallintestine. Thus, the film coating (which surrounds the drug reservoir)should be poorly permeable for the drug upon exposure to mediasimulating the contents of these organs (in order to avoid prematuredrug release and subsequent absorption into the blood stream). If apolymeric film coating takes up significant amounts of water or loosesconsiderable amounts of dry mass upon exposure to a bulk fluid, itspermeability for drug molecules can be expected to remarkably increase []. For this reason, the water uptake and dry mass loss kinetics of thinpeas starch: ethylcellulose films were monitored upon exposure to: (a)0.1 M HCl (simulating the contents of the stomach) for 2 h, and (b)phosphate buffer pH 6.8 (simulating the contents of the small intestine)for 8 h. FIG. 14 shows the experimentally determined water contents ofthe films as a function of time. The peas starch:ethylcellulose blendratio was varied from 1:2 to 1:4, as indicated. For reasons ofcomparison, also films consisting only of (plasticized) ethylcellulosewere studied (filled triangles). As it can be seen, the water uptakerates and extents increased with increasing peas starch contents, due tothe hydrophilic nature of this polymer. Importantly, the water uptakeremains limited in all cases (below 30%). The slightly higher wateruptake rates and extents at pH 6.8 compared to pH 1.2 (FIG. 14 b versus14a) can probably be attributed to the presence of sodium dodecylsulfate (SDS), which is present in the aqueous ethylcellulose dispersionused for film preparation, serving as a stabilizer of this dispersion.At low pH, SDS is protonated and non-charged, whereas at pH 6.8 it isdeprotonated and, thus, negatively charged. Hence, its hydrophilicity isincreased and water penetration into the films is facilitated [ ].

FIG. 15 shows the experimentally measured dry mass loss kinetics ofvarious peas starch: ethylcellulose films upon exposure to: (a) 0.1 MHCl, and (b) phosphate buffer pH 6.8. As it can be seen, the dry massloss rate and extent slightly increased with increasing peas starchcontent, because this polysaccharide significantly swells upon contactwith water and, thus, facilitates the leaching of water-soluble filmcompounds (e.g., of the water-soluble plasticizer TEC [ ]) into thesurrounding bulk fluid. The lowest mass loss was observed with peasstarch-free films, irrespective of the type of medium. This can beattributed to the fact that ethylcellulose is poorly swellable andpermeable upon contact with aqueous media. It effectively hinders theleaching of water-soluble compounds.

Thus, the observed water uptake and dry mass loss kinetics of peasstarch: ethylcellulose films are very promising with respect to thepotential use of these films as barrier membranes hindering drug releasein stomach and small intestine. If required, the film thickness and/orethylcellulose contents might be increased. However, care should betaken that sufficient amounts of peas starch are present in thecoatings, because this compound is intended to induce the onset of drugrelease in the colon (being degraded by enzymes secreted from colonicbacteria.

B.2. Mechanical Properties of Thin Films

In addition to limited water uptake and dry mass loss, polymeric filmcoatings aiming at site specific drug delivery to the colon shouldprovide a sufficient mechanical stability: Due to the physiologicalmotility of the stomach and small intestine, mechanical stress isexerted onto the coated dosage forms. If the film coatings are fragile,crack formation occurs and the drug is rapidly released throughwater-filled channels. To evaluate the mechanical stability of theinvestigated peas starch: ethylcellulose blends, a texture analyzer andthe puncture test were used. FIG. 16 shows the: (a) puncture strength atbreak, (b) % elongation at break, and (c) energy required to break thinpolymeric films in the dry state. Clearly, the mechanical stability ofthe films significantly increased with increasing ethylcellulosecontent. Interestingly, all values are relatively high, suggesting thatfilm coatings with a common thickness can withstand the mechanicalstress experienced within the gastro intestinal tract in vivo.

However, it has to be pointed out that the results shown in FIG. 16 wereobtained with dry films. Upon contact with aqueous bulk fluids, themechanical properties of a polymeric film coating can significantlychange, for instance due to compound leaching into the surrounding bulkfluid and/or the plasticizing effect of water [Erreur ! Signet nondéfini.,Erreur ! Signet non défini.]. For these reasons, the mechanicalproperties of the investigated peas starch: ethylcellulose films werealso measured upon up to 2 h exposure to 0.1 M HCl and up to 8 hexposure to phosphate buffer pH 6.8. FIGS. 17 a and 17 b show therespective energies required to break the wet films of differentcomposition. Clearly, the mechanical strength of all films decreasedwith increasing exposure time, irrespective of the type of bulk fluid.This can at least partially be attributed to the leaching of thewater-soluble plasticizer TEC into the bulk fluids [Erreur ! Signet nondéfini.]. As expected, an increase in the ethylcellulose contentresulted in increased energies required to break the films in bothmedia. Importantly, the observed values suggest that all film coatingsare likely to withstand the mechanical stress encountered in vivo withinthe gastro intestinal tract, also in the wet state (at commonly usedcoating levels).

B.3. Drug Release in the Upper Gastro Intestinal Tract

Ideally, no or very little drug should be released from the dosage formin the stomach and small intestine. The solid curves in FIG. 18 show theexperimentally determined drug release kinetics from pellets coated withpeas starch:ethylcellulose 1:2 at a coating level of 0, 5, 10, 15 and20% (w/w) into: 0.1 M HCl (for 2 h), followed by phosphate buffer pH 6.8(for 9 h) at 37° C. As it can be seen, 5-amino salicylic acid wasrapidly released from uncoated pellets as well as from pellets coatedwith only 5% peas starch:ethylcellulose 1:2. This can at least partiallybe attributed to the water uptake and dry mass loss of these filmcoatings upon exposure to the release media (FIGS. 14 and 15), incombination with an insufficient thickness of the polymeric barrier.Importantly, at coating levels equal to and above 10% (w/w), drugrelease was effectively slowed down (probably due to the increase in thelength of the diffusion pathways and increased mechanical stability ofthe film coatings).

However, it has to be pointed out that the presence of enzymes withinthe gastro intestinal tract in vivo might significantly affect the filmcoating properties, e.g. due to partial polymer degradation. For thisreason, drug release from the coated pellets was also measured in: (i)0.1 M HCl containing 0.32% pepsin (for 2 h), followed by (ii) phosphatebuffer pH 6.8 containing 1% pancreatin (for 9 h). The respective resultsare indicated by the dotted curves in FIG. 18. Clearly, in all cases thedrug release rate only slightly increased. Thus, the importance of suchenzymatic degradation in vivo is likely to be limited.

As an increase in the relative ethylcellulose contents of the filmsresulted in decreased water uptake and dry mass loss rates and extents(FIGS. 14 and 15) as well as increased mechanical stability of the filmsin the dry and wet state (FIGS. 16 and 17), drug release was alsomeasured from pellets coated with peas starch:ethylcellulose 1:3, 1:4and 1:5 blends at different coating levels (FIG. 19). Interestingly, inthese cases even a film coating of only 5% (w/w) is able to slow downdrug release. However, coating levels of 10% or more are moreappropriate, because drug release is almost completely suppressed withinthe observation period.

Based on the obtained results (FIGS. 14-19), pellets coated with peasstarch:ethylcellulose 1:4 at a coating level of 15 and 20% have beenselected for further studies.

B.4. Drug Release in the Entire Gastro Intestinal Tract

Once the dosage form reaches the colon, the film coating should becomepermeable for the drug and release the latter in a time-controlledmanner. FIG. 20 a shows the experimentally measured release of 5-aminosalicylic acid from pellets coated with peas starch:ethylcellulose 1:4at a coating level of 15 and 20% (w/w) into: (i) 0.1 M HCl for 2 h,followed by (ii) phosphate buffer pH 6.8 for 9 h, and (c) culture mediuminoculated with fecal samples from inflammatory bowel disease patientsfor 10 h (solid curves). For reasons of comparison, also drug releaseupon exposure to culture medium free of feces is illustrated (dottedcurves). Clearly, drug release set on as soon as the pellets came intocontact with fecal samples. This can be attributed to the (at leastpartial) degradation of peas starch by the enzymes secreted by thebacteria present in the colon of the patients. The decrease in polymermolecular weight and subsequent diffusion of degradation products intothe surrounding bulk fluids renders the remaining macromolecular networkmore mobile. Consequently, also the mobility of the drug moleculeswithin the film coating increases and, thus, the release rate increases.In contrast, the drug release rate remained low upon exposure to culturemedium free of feces (dotted curves in FIG. 20 a). This confirms thatdrug release is triggered by the enzymes present in the colon of IBDpatients. From a practical point of view, a coating level of 15% seemsto be preferable to a coating level of 20% (potentially resulting in toolow drug release in the colon).

As the regular supply of fresh fecal samples from inflammatory boweldisease patients is difficult to assure (and since the samples cannot bedeep-frozen or freeze-dried without significant damage of themicroflora), it is highly desirable to provide an alternative type ofrelease medium, simulating the conditions in the colon of a patient. Fordrug delivery systems that are sensitive to the presence of bacterialenzymes, caution has to be paid that the bulk fluid contains the crucialtypes and amounts of bacteria. In this study, culture medium inoculatedwith Bifidobacterium has been tested as potential alternative to culturemedium inoculated with fresh fecal samples. FIG. 20 b shows the observeddrug release rate from the same types of pellets as shown in FIG. 20 aupon exposure to: 0.1 M HCl (for 2 h), phosphate buffer pH 6.8 (for 9 h)and culture medium inoculated with Bifidobacterium (for 10 h). ComparingFIGS. 20 a and 20 b, it becomes obvious that culture medium inoculatedwith Bifidobacterium shows a promising potential as substitute for freshfecal samples from IBD patients, in particular for routine applications(such as quality controls during large scale production).

Similar results were obtained using dibutyl sebacate as plasticizer(data no shown) confirming that the efficiency of pea starch incontrolled released delivery is not plasticizer dependent.

B.5. Storage Stability

A very important aspect from a practical point of view is the long termstability of a controlled drug delivery system. Dosage forms shouldideally be stable during at least 3 years. In case of polymer coateddelivery systems the resulting drug release rate might eventuallyincrease with increasing storage time, e.g. due to drug migration intothe film coating. FIG. 21 shows the drug release kinetics from pelletscoated with peas starch:ethylcellulose 1:4 at a coating level of 10, 15and 15% (as indicated) before and after 1 year storage in open glassvials (solid and dotted curves). The systems were exposed to 0.1 M HClfor 2 h, and subsequently to phosphate buffer pH 6.8 for 9 h. As it canbe seen, the release rate remained unaltered during long term storage.The same is true for pellets coated with peas starch:ethylcellulose 1:2,1:3 and 1:5 at a coating level of 10, 15 and 20% (data not shown). Thus,the proposed drug delivery systems are long term stable.

C. Conclusions

Peas starch: ethylcellulose-based film coatings have been proposed witha highly promising potential for site specific drug delivery to thecolon: Drug release from coated pellets can effectively be suppressed inmedia simulating the contents of the stomach and small intestine. Butonce the devices come into contact with fecal samples, drug release setson and is time-controlled, due to the partial degradation of the peasstarch by enzymes secreted from bacteria present in the colon ofinflammatory bowel disease patients. Thus, this type of advanceddelivery systems allows avoiding premature drug release in the uppergastro intestinal tract (and subsequent absorption into the bloodstream), whiling assuring that the drug is released at the site ofaction. Consequently, undesired side effects in the rest of the humanbody can be expected to be minimized, while the therapeutic effects ofthe drug are likely to be optimized.

Example 4

A. Materials and Methods

A.1 Materials

2,4,6-Trinitrobenzene sulfonic acid (TNBS) (Sigma-Aldrich, Isle d'AbeauChesnes, France); cysteinated Ringer solution (Merck, Darmstadt,Germany); BMD (NUTRIOSE® FB 06; Roquette Freres, Lestrem, France); Peasstarch N-735 (peas starch; Roquette Freres, Lestrem, France); aqueousethylcellulose dispersion (Aquacoat ECD 30; FMC Biopolymer,Philadelphia, USA); triethylcitrate (TEC; Morflex, Greensboro, USA);5-aminosalicylic acid (5-ASA; Sigma-Aldrich, Isle d'Abeau Chesnes,France); microcrystalline cellulose (Avicel PH 101; FMC Biopolymer,Brussels, Belgium); polyvinylpyrrolidone (PVP, Povidone K 30)(Cooperation Pharmaceutique Francaise, Melun, France); Pentasa® (coatedpellets, Ferring, batch number: JX 155), Asacol® (coated granules,Meduna, batch number: TX 143).

A.2 Preparation of Bmd: Ethylcellulose and Peas Starch: EthylcelluloseCoated Pellets

5-Amino salicylic acid (5-ASA) loaded pellet starter cores (diameter:0.7-1.0 mm; 60% 5-ASA, 32% microcrystalline cellulose, 4% bentonite, 4%PVP) were prepared by extrusion and subsequent spheronisation asfollows: The respective powders were blended in a high speed granulator(Gral 10; Collette, Antwerp, Belgium) and purified water was added untila homogeneous mass was obtained (41 g of water for 100 g of powderblend). The wetted mixture was passed through a cylinder extruder (SKM/R, holes: 1 mm diameter, 3 mm thickness, rotation speed: 96 rpm;Alexanderwerk, Remscheid, Germany). The extrudates were subsequentlyspheronised at 520 rpm for 2 min (Spheroniser Model 15; Calveva, Dorset,UK) and dried in a fluidized bed (ST 15; Aeromatic, Muttenz,Switzerland) at 40° C. for 30 min. The size fraction 0.7-1.0 mm wasobtained by sieving.

The obtained drug loaded starter cores were subsequently coated in afluidized bed coater, equipped with a Wurster insert (Strea 1;Aeromatic-Fielder, Bubendorf, Switzerland) with BMD:ethylcellulose 1:4blends (BMD:EC coated pellets) or with peas starch: ethylcellulose 1:2blends (peas starch:EC coated pellets) until a weight gain of 15% (w/w)(BMD:EC coated pellets) or 20% (w/w) (peas starch: EC coated pellets)was achieved.

BMD was dissolved in purified water (5% w/w), blended with plasticizedaqueous ethylcellulose dispersion (25% TEC, overnight stirring; 15% w/wpolymer content) at a ratio of 1:4 (w/w, based on the non-plasticizedpolymer dry mass) and stirred for 6 h prior to coating. The drug-loadedpellet cores were coated in a fluidized bed coater equipped with aWurster insert (Strea 1; Aeromatic-Fielder, Bubendorf, Switzerland)until a weight gain of 15% (w/w) was achieved. The process parameterswere as follows: inlet temperature=39±2° C., product temperature=40±2°C., spray rate=1.5−3 g/min, atomization pressure=1.2 bar, nozzlediameter=1.2 mm. After coating, the beads were further fluidized for 10min and subsequently cured in an oven for 24 h at 60° C.

Peas starch was dispersed in purified water at 65-75° C. (5% w/w).Aqueous ethylcellulose dispersion (15% w/w solids content) wasplasticized for 24 h with 25% TEC (w/w, referred to the solids contentof the dispersion). The peas starch and ethylcellulose dispersions wereblended at room temperature at the following ratio: 1:2(polymer:polymer, w:w). The mixture was stirred for 6 h prior tocoating. The drug-loaded pellet cores were coated in a fluidized bedcoater equipped with a Wurster insert (Strea 1; Aeromatic-Fielder,Bubendorf, Switzerland) until a weight gain of 20% (w/w) was achieved.The process parameters were as follows: inlet temperature=39±2° C.,product temperature=40±2° C., spray rat=1.5−3 g/min, atomizationpressure=1.2 bar, nozzle diameter=1.2 mm. Afterwards, the pellets werefurther fluidized for 10 min and subsequently cured in an oven for 24 hat 60° C.

A.3 Induction of Colitis and Study Design

Male Wistar rats (250 g) were used for the in vivo study, which wasconducted in accredited establishment at the Institut Pasteur de Lille(A 35009), according to governmental guidelines (86/609/CEE). Fouranimals were housed per cage, all rats had free access to tap water.

At the beginning of the experiment (day 0), the rats were divided in sixgroups (5-8 animals/group). Two groups received standard chow (negativeand positive control groups). The other groups received food with eitherPentasa® pellets (n=8), Asacol® pellets (n=8), BMD: ethylcellulosecoated pellets (n=8) or peas starch: ethylcellulose coated pellets(n=8). These four different chows were prepared using the “food admix”technique. All systems were added to obtain a dose of 5-ASA of 150mg/kg/day.

At day 3, colitis was induced as follows: The rats were anesthetized for90-120 min using pentobarbital (40 mg/kg) and received an intrarectaladministration of TNBS (250 μl, 20 mg/rat) dissolved in a 1:1 mixture ofan aqueous 0.9% NaCl solution with 100% ethanol. Control rats (negativecontrol) received an intrarectal administration of the vehicle only (1:1mixture of an aqueous 0.9% NaCl solution with 100% ethanol). Animalswere sacrificed 3 days after intrarectal TNBS or vehicle administration(day 6).

A.4 Macroscopic and Histological Assessment of Colitis

Macroscopic and histological indications of colitis were evaluatedblindly by two investigators. A colon specimen located precisely 4 cmabove the anal canal was used for histological evaluation according tothe Ameho criteria. This grading on a scale from 0 to takes into accountthe degree of inflammation infiltrate, the presence of erosion,ulceration, or necrosis, and the depth and surface extension of lesions.

A.5 Statistics

All comparisons were analyzed using the nonparametric test(Mann-Whitney) test. Differences were judged statistically significantif the P value was <0.05.

B Results and Discussion

TNBS-induced colitis is improved by the treatment with BMD:ethylcellulose coated pellets and peas starch: ethylcellulose coatedpellets.

The development of colitis in animals subjected to intrarectal TNBSadministration was characterized. Control rats (negative control group),sacrificed 3 days after intrarectal administration of the vehicle only(a 1:1 mixture of an aqueous 0.9% NaCl solution with 100% ethanol), hadno macroscopic lesions in the colon (FIG. 22A). In contrast, a severecolitis was induced as early as 3 d after administration of TNBS (FIG.22B). On the histological level, no abnormalities were detected incontrol rats (FIG. 24: control=negative control group). In contrast, 3days after the administrations of TNBS, colon histology wascharacterized by large areas of ulceration with a neutrophilicinfiltrate, necrosis extending deeply into the muscular layer (FIG. 24:TNBS). The colon of animals treated with BMD: ethylcellulose coatedpellets showed a significant reduction of the lesion (FIG. 22). Theresults were similar for rats treated with peas starch: ethylcelullosecoated pellets (data not shown). Furthermore, the effects of treatmentswith Pentasa® pellets, Asacol® pellets, BMD: ethylcellulose coatedpellets and peas starch: ethylcellulose coated pellets on TNBS-inducedcolon lesions were studied using the Ameho score (FIG. 29). Untreatedrats with colitis were investigated for reasons of comparison. Optimaleffects were obtained with BMD: ethylcellulose coated pellets and withpeas starch: ethylcellulose coated pellets. Three days after inductionof colitis, a significant decrease in the macroscopic lesion score wasobserved in rats that had received BMD: ethylcellulose coated pelletsand peas starch: ethylcellulose coated pellets preventively comparedwith untreated rats with colitis. Parallel to the macroscopicinflammation, histological analysis also confirmed major differencesbetween animals treated with: (i) TNBS intrarectally, (ii) TNBSintrarectally and Pentasa® pellets orally, (iii) TNBS intrarectally andAsacol® pellets orally, (v) TNBS intrarectally and BMD: ethylcellulosecoated pellets orally, and (v) TNBS intrarectally and peas starch:ethylcellulose coated pellets orally (FIG. 24). This was reflected by asignificant decrease of the Ameho inflammation score at 3 d after TNBSadministration (FIG. 23). Clearly, the administration of BMD:ethylcellulose coated pellets and of peas starch: ethylcellulose coatedpellets reduced the inflammatory lesions which consisted of smallerpolymorphic inflammatory infiltrates, limited edema and small focalnecrosis lesions (FIG. 24). Thickening of the colon wall, with apredominant inflammatory infiltrate in the lamina propria, and necrosisextending deeply into the muscular and serosal layer are evident in thecase of treatment with TNBS, TNBS and Pentasa® pellets and TNBS andAsacol® pellets.

These results clearly prove the efficacy of the proposed novel filmcoatings for colon targeting in vivo.

TABLE 1 Healthy Crohn's Ulcerative subjects Disease Colitis Number 10 115 Mean age 40 +/− 15 32 +/− 12 36 +/− 20 Mean total counts 9.88 +/− 0.489.15 +/− 1.30 9.88 +/− 0.57 [log UFC/g] Number of strains 28 34 14 Mean2.8 3.1 2.8 Anaerobes Bacteroides 9 10 3 Prevotella 2 2 2 Fusobacterium3 3 2 Veillonella 0 0 1 Clostridium 0 5 1 Bifidobacterium 9 3 1 OtherGram + rods 3 2 2 Gram + cocci 1 2 0 Aerobes Enterobacteria 1 3 2Escherichia coli 1 2 1 Citrobacter freundii 0 2 1 Lactobacillus 0 2 0Streptococcus 0 2 0 Mean counts McConkey 6.30 +/− 1.19 7.16 +/− 1.488.01 +/− 1.06 agar Number of strains 10 14 8 Escherichia coli 10 6 4 E.coli lac- 0 1 0 Citrobacter freundii 0 3 1 Klebsiella pneumoniae 0 1 1Klebsiella oxytoca 0 2 0 Enterobacter cloacae 0 1 0 Other Gram - rods 00 1

TABLE 2 Puncture Elongation Energy at Blend strength ± at break ± break± (s), ratio (s), MPa (s), % MJ/m³ MD 1:2 0.34 ± (0.05) 0.43 ± (0.08)0.012 ± (0.005) 1:3 0.36 ± (0.09) 0.57 ± (0.05) 0.014 ± (0.006) 1:4 0.43± (0.07) 0.53 ± (0.04) 0.011 ± (0.003) 1:5 0.42 ± (0.11) 0.58 ± (0.07)0.015 ± (0.009) PS HP-PG 1:2 0.45 ± (0.04) 0.55 ± (0.09) 0.016 ± (0.008)1:3 0.40 ± (0.03) 0.53 ± (0.07) 0.012 ± (0.007) 1:4 0.42 ± (0.09) 0.60 ±(0.09) 0.016 ± (0.008) 1:5 0.50 ± (0.08) 0.60 ± (0.05) 0.020 ± (0.004)MS7 A-PG 1:2 0.78 ± (0.09) 0.63 ± (0.02) 0.061 ± (0.005) 1:3 0.84 ±(0.05) 0.67 ± (0.08) 0.065 ± (0.009) 1:4 0.85 ± (0.04) 0.66 ± (0.07)0.070 ± (0.011) 1:5 0.87 ± (0.05) 0.75 ± (0.02) 0.073 ± (0.006) MS6 A-PG1:2 0.60 ± (0.01) 0.50 ± (0.07) 0.052 ± (0.002) 1:3 0.52 ± (0.05) 0.75 ±(0.10) 0.068 ± (0.008) 1:4 0.76 ± (0.02) 0.82 ± (0.04) 0.077 ± (0.006)1:5 0.77 ± (0.03) 0.81 ± (0.06) 0.075 ± (0.010) MS6 HP-PG 1:2 0.53 ±(0.07) 0.72 ± (0.05) 0.053 ± (0.010) 1:3 0.64 ± (0.03) 0.81 ± (0.07)0.066 ± (0.009) 1:4 0.63 ± (0.02) 0.82 ± (0.07) 0.062 ± (0.009) 1:5 0.87± (0.03) 0.77 ± (0.05) 0.070 ± (0.010)

TABLE 3 Simulated GI Exposure segment time Release medium pH Stomach   2h 0.1M HCl 1.2 Duodenum 0.5 h Phosphate buffer 5.5 (Eur. Pharm. 5)Jejunum-Ileum   9 h Phosphate buffer 6.8 (USP 30) Caecum 0.5 h Phosphatebuffer 6.0 (USP 30) Proximal Colon   6 h Phosphate buffer 7.0 (USP 30)Distal Colon  18 h Phosphate buffer 7.4 (USP 30)

1. A controlled release delivery dosage form for controlled release ofan active ingredient, comprising an active ingredient coated in apolymeric mixture of: at least a water insoluble polymer and a starchcomposition comprising one component selected from the group consistingof a starch having an amylose content of between 25 and 45%, a modifiedstarch having an amylose content of between 50 and 80% and a legumestarch.
 2. The controlled release delivery dosage form according toclaim 1, comprising a core, the active ingredient being dispersed ordissolved in the core.
 3. The controlled release delivery dosage formaccording to claim 1, wherein the starch composition: water insolublepolymer ratio is between 1:2 and 1:8.
 4. The controlled release deliverydosage form according to claim 1, wherein the starch compositionexhibits an amylose content of between 25 and 45%, this percentage beingexpressed by dry weight with respect to the dry weight of starch presentin said composition.
 5. The controlled release delivery dosage formaccording to claim 1, wherein the starch composition comprises at leastone legume or cereal starch.
 6. The controlled release delivery dosageform according to claim 1, wherein the starch composition comprises atleast one modified starch, said modified starch being stabilized.
 7. Thecontrolled release delivery dosage form according to claim 2, whereinthe core has a coating level of 5% to 30%.
 8. The controlled releasedelivery dosage form according to claim 7, wherein the core has acoating level of 10% to 20%.
 9. The controlled release delivery dosageform according to claim 1, wherein the polymeric mixture comprises aplasticizer, preferably in a content between 25% to 30% w/w referred tothe water insoluble polymer content.
 10. The controlled release deliverydosage form according to claim 1, wherein the water insoluble polymer isselected from the group consisting of acrylic and/or methacrylic esterpolymers, polymers or copolymers of acrylate or methacrylate polyvinylesters, polyvinyl acetates, polyacrylic acid esters, and butadienestyrene copolymers methacrylate ester copolymers, ethyl cellulose,cellulose acetate phtalate, polyvinyl acetate phtalate, shellac,methacrylic acid copolymers, cellulose acetate trimellitate,hydroxypropyl methylcellulose phtalate, zein, starch acetate.
 11. Thecontrolled release delivery dosage form according to claim 9, whereinthe plasticizer is a water soluble plasticizer, the plasticizer beingpreferably selected from the group consisting of polyols, organicesters, oils or glycerides, soya lecithin, alone or as a mixture withone another.
 12. The controlled release delivery dosage form accordingto, wherein said controlled release delivery dosage form is amultiparticulate dosage form.
 13. A method for preparing a controlledrelease delivery dosage form for controlled release of an activeingredient in the colon of patients having a colonic microfloraimbalance or in the colon of healthy subjects, as claimed in claim 1,said method comprising: forming a polymeric mixture of: at least onewater insoluble polymer and a starch composition comprising at least onecomponent selected from the group consisting of a starch having anamylose content of between 25 and 45%, a modified starch having anamylose content of between 50 and 80% and a legume starch, coating saidactive ingredient in the polymeric mixture.